VENTANA MMR IHC Panel Interpretation Guide for Staining of Colorectal Tissue

VENTANA MMR IHC Panel Interpretation Guide for Staining of Colorectal Tissue VENTANA anti-mlh1 (M1) Mouse Monoclonal Primary Antibody VENTANA anti-pms2 (A16-4) Mouse Monoclonal Primary Antibody VENTANA anti-msh2 (G219-1129) Mouse Monoclonal Primary Antibody VENTANA anti-msh6 (SP93) Rabbit Monoclonal Primary Antibody VENTANA anti-braf V600E (VE1) Mouse Monoclonal Primary Antibody

Table of Contents Introduction... 4 Colorectal Cancer and Lynch Syndrome... 4 Stratification for Lynch Syndrome in CRC... 4 VENTANA MMR IHC Panel... 5 Use of the VENTANA MMR IHC Panel to identify MMR Status and Probable Lynch Syndrome... 5 VENTANA MMR IHC Panel... 6 Intended Use of VENTANA MMR IHC Panel... 6 VENTANA anti-mlh1 (M1) Mouse Monoclonal Primary Antibody... 6 VENTANA anti-pms2 (A16-4) Mouse Monoclonal Primary Antibody... 6 VENTANA anti-msh2 (G219-1129) Mouse Monoclonal Primary Antibody... 7 VENTANA anti-msh6 (SP93) Rabbit Monoclonal Primary Antibody... 7 VENTANA anti-braf V600E (VE1) Mouse Monoclonal Primary Antibody... 8 Purpose of Interpretation Guide... 8 Specimen Flow for Staining with the VENTANA MMR IHC Panel... 9 Clinical Evaluation of MMR IHC Assays... 10 Evaluating Staining Patterns and Intensities... 10 Specimen Flow for MMR IHC Assays... 11 Morphology and Background Acceptability Criteria... 12 System-Level Control... 12 Internal Positive Controls... 14 CRC Case Criteria for Clinical Evaluation... 15 Scoring Algorithm for MMR IHC Assays... 16 Decision Tree for MMR IHC Assays... 17 Cases with Intact MMR Status... 18 CRC Cases with Intact MMR protein expression... 18 Cases with MMR Status of Loss... 22 CRC Cases with loss of MMR protein expression... 22 Challenging Cases... 26 Non-specific background staining... 27 Focal Staining... 29 Punctate Staining... 30 Clinical Evaluation of VENTANA anti-braf V600E IHC Assay... 31 Evaluating Staining Patterns and Intensities... 31 Specimen Flow for VENTANA anti-braf V600E IHC Assay... 32 Morphology and Background Acceptability Criteria... 33 System-level Control... 33 Scoring Algorithm for the VENTANA anti-braf V600E IHC Assay... 34 Decision Tree... 35 Cases Negative for BRAF V600E... 36 CRC Cases with BRAF V600E Negative Status... 36 Cases Positive for BRAF V600E... 38 CRC Cases with BRAF V600E Positive Status... 38 Challenging Cases... 42 Non-Specific Background Staining... 43 1016703EN Interpretation Guide 2017 2

Non-Specific High Background Staining... 44 Nuclear Staining... 45 Focal Staining... 47 VENTANA MMR IHC Panel Case Examples... 48 Cut Slide Stability... 52 References... 53 1016703EN Interpretation Guide 2017 3

Introduction Colorectal Cancer and Lynch Syndrome Colorectal cancer (CRC) is the third most common cancer in men, and the second in women, with an estimated 1.4 million new cases and 694,000 deaths occurring worldwide in 2012. Incidence rates vary widely by geographic location, with the highest estimated rates in Australia/New Zealand, Europe, and North America. 1 In the United States alone CRC represents 8.0% of all new cancer cases and an estimated 1.2 million people were living with colon and rectum cancer as of 2012. 2 The risk of developing CRC is influenced by both environmental factors (e.g., dietary factors, obesity, smoking and alcohol use) and genetic factors. While the majority of CRC cases are sporadic in nature, 5-10% of cases are due to inherited autosomal dominant mutations. 3 The most common subtype of hereditary CRC is Lynch syndrome, which accounts for 3% of all CRC diagnoses. 4 Lynch syndrome (Hereditary Non-Polyposis Colon Cancer, HNPCC), was described in the 1960s and identified a link between the loss of DNA mismatch repair (MMR) function and cancer. 5 Loss of any MMR protein (MLH1, PMS2, MSH2 or MSH6) may lead to microsatellite instability and a higher risk of not only colorectal cancer, but also cancer of stomach, brain, skin and, in women, endometrium and ovaries. Patients with Lynch syndrome have a 40-60% lifetime risk for colorectal cancer. 6 Stratification for Lynch Syndrome in CRC Using IHC assays for MLH1, PMS2, MSH2, and MSH6, the MMR status of the tumor may be determined. Detection of all four proteins in the tumor indicates normal or proficient mismatch repair status (pmmr). Loss of MLH1 expression is almost invariably accompanied with the loss of its heterodimer partner, PMS2. In sporadic occurrences of CRC, expression of the MLH1 gene may be suppressed by methylation of its promoter. If the result indicates a loss of MLH1 protein, testing to see if the BRAF V600E mutation is present will stratify the tumor as sporadic or probable Lynch syndrome and indicate the need for additional testing of the MLH1 promoter methylation state. The presence of the BRAF V600E mutation with loss of MLH1 protein indicates the tumor is the result of a sporadic occurrence and makes Lynch syndrome as the underlying cause of malignancy highly unlikely. 5 If BRAF V600E mutation and MLH1 promoter methylation is negative, the deficient DNA mismatch repair (dmmr) status is consistent with Lynch syndrome and warrants genetic testing for a confirmatory diagnosis. 5 1016703EN Interpretation Guide 2017 4

The use of IHC for the detection of PMS2, MSH2 and MSH6 proteins is a more direct indicator of germline mutational status. The loss of PMS2, in the presence of MLH1 expression, or loss of MSH2 or MSH6 expression designates the tumor as dmmr and is consistent with Lynch syndrome. All individuals with suspected Lynch syndrome would be referred for genetic counseling and further genetic testing to confirm the presence of the suspected mutation. VENTANA MMR IHC Panel Use of the VENTANA MMR IHC Panel to identify MMR Status and Probable Lynch Syndrome The VENTANA MMR IHC Panel (VENTANA anti-mlh1 (M1) Mouse Monoclonal Primary Antibody, VENTANA anti- PMS2 (A16-4) Mouse Monoclonal Primary Antibody, VENTANA anti-msh2 (G219-1129) Mouse Monoclonal Primary Antibody, VENTANA anti-msh6 (SP93) Rabbit Monoclonal Primary Antibody and VENTANA anti-braf V600E (VE1) Mouse Monoclonal Primary Antibody) aids in the identification of patients with a deficiency in MMR and the stratification of colorectal cancer (CRC) as sporadic or probable Lynch syndrome. This is summarized in Figure 1. Figure 1: Use of VENTANA MMR IHC Panel 1016703EN Interpretation Guide 2017 5

VENTANA MMR IHC Panel Intended Use of VENTANA MMR IHC Panel VENTANA anti-mlh1 (M1) Mouse Monoclonal Primary Antibody VENTANA anti-mlh1 (M1) Mouse Monoclonal Primary Antibody (VENTANA anti-mlh1 (M1) antibody) is intended for the qualitative detection of MLH1 protein in formalin-fixed, paraffin-embedded tissue sections. VENTANA anti-mlh1 (M1) antibody is ready to use on BenchMark ULTRA, XT and GX instruments with the OptiView DAB IHC Detection Kit and ancillary reagents. VENTANA anti-mlh1 (M1) antibody is part of the VENTANA MMR IHC Panel which includes VENTANA anti-pms2 (A16-4) Mouse Monoclonal Primary Antibody, VENTANA anti-msh2 (G219-1129) Mouse Monoclonal Primary Antibody, VENTANA anti-msh6 (SP93) Rabbit Monoclonal Primary Antibody and VENTANA anti-braf V600E (VE1) Mouse Monoclonal Primary Antibody. The VENTANA MMR IHC Panel is indicated for the detection of mismatch repair protein deficiency as a test for the identification of individuals at risk for Lynch syndrome in patients diagnosed with colorectal cancer (CRC), and, with BRAF V600E status, as an aid to differentiate between sporadic and probable Lynch syndrome CRC in the absence of MLH1 protein expression. These products should be interpreted by a qualified pathologist in conjunction with histological examination, relevant clinical information, and proper controls. Intended for in vitro diagnostic (IVD) use. VENTANA anti-pms2 (A16-4) Mouse Monoclonal Primary Antibody VENTANA anti-pms2 (A16-4) Mouse Monoclonal Primary Antibody (VENTANA anti-pms2 (A16-4) antibody) is intended for the qualitative detection of PMS2 protein in formalin-fixed, paraffin-embedded tissue sections. VENTANA anti-pms2 (A16-4) antibody is ready to use on BenchMark ULTRA, XT and GX instruments with the OptiView DAB IHC Detection Kit, OptiView Amplification Kit and ancillary reagents. VENTANA anti-pms2 (A16-4) antibody is part of the VENTANA MMR IHC Panel which includes VENTANA anti-mlh1 (M1) Mouse Monoclonal Primary Antibody, VENTANA anti-msh2 (G219-1129) Mouse Monoclonal Primary Antibody, VENTANA anti-msh6 (SP93) Rabbit Monoclonal Primary Antibody and VENTANA anti-braf V600E (VE1) Mouse Monoclonal Primary Antibody. The VENTANA MMR IHC Panel is indicated for the detection of mismatch repair protein deficiency as a test for the identification of individuals at risk for Lynch syndrome in patients diagnosed with colorectal cancer (CRC), and, with BRAF V600E status, as an aid to differentiate between sporadic and probable Lynch syndrome CRC in the absence of MLH1 protein expression. These products should be interpreted by a qualified pathologist in conjunction with histological examination, relevant clinical information, and proper controls. Intended for in vitro diagnostic (IVD) use. 1016703EN Interpretation Guide 2017 6

VENTANA anti-msh2 (G219-1129) Mouse Monoclonal Primary Antibody VENTANA anti-msh2 (G219-1129) Mouse Monoclonal Primary Antibody (VENTANA anti-msh2 (G219-1129) antibody) is intended for the qualitative detection of MSH2 protein in formalin-fixed, paraffin-embedded tissue sections. VENTANA anti-msh2 (G219-1129) antibody is ready to use on BenchMark ULTRA, XT and GX instruments with the OptiView DAB IHC Detection Kit and ancillary reagents. VENTANA anti-msh2 (G219-1129) antibody is part of the VENTANA MMR IHC Panel which includes VENTANA anti- MLH1 (M1) Mouse Monoclonal Primary Antibody, VENTANA anti-pms2 (A16-4) Mouse Monoclonal Primary Antibody, VENTANA anti-msh6 (SP93) Rabbit Monoclonal Primary Antibody and VENTANA anti-braf V600E (VE1) Mouse Monoclonal Primary Antibody. The VENTANA MMR IHC Panel is indicated for the detection of mismatch repair protein deficiency as a test for the identification of individuals at risk for Lynch syndrome in patients diagnosed with colorectal cancer (CRC), and, with BRAF V600E status, as an aid to differentiate between sporadic and probable Lynch syndrome CRC in the absence of MLH1 protein expression. These products should be interpreted by a qualified pathologist in conjunction with histological examination, relevant clinical information, and proper controls. Intended for in vitro diagnostic (IVD) use. VENTANA anti-msh6 (SP93) Rabbit Monoclonal Primary Antibody VENTANA anti-msh6 (SP93) Rabbit Monoclonal Primary Antibody (VENTANA anti-msh6 (SP93) antibody) is intended for the qualitative detection of MSH6 protein in formalin-fixed, paraffin-embedded tissue sections. VENTANA anti- anti- MSH6 (SP93) antibody is ready to use on BenchMark ULTRA, XT and GX instruments with the OptiView DAB IHC Detection Kit and ancillary reagents. VENTANA anti-msh6 (SP93) antibody is part of the VENTANA MMR IHC Panel which includes VENTANA anti-mlh1 (M1) Mouse Monoclonal Primary Antibody, VENTANA anti-pms2 (A16-4) Mouse Monoclonal Primary Antibody, VENTANA anti-msh2 (G219-1129) Mouse Monoclonal Primary Antibody and VENTANA anti-braf V600E (VE1) Mouse Monoclonal Primary Antibody. The VENTANA MMR IHC Panel is indicated for the detection of mismatch repair protein deficiency as a test for the identification of individuals at risk for Lynch syndrome in patients diagnosed with colorectal cancer (CRC), and, with BRAF V600E status, as an aid to differentiate between sporadic and probable Lynch syndrome CRC in the absence of MLH1 protein expression. These products should be interpreted by a qualified pathologist in conjunction with histological examination, relevant clinical information, and proper controls. Intended for in vitro diagnostic (IVD) use. 1016703EN Interpretation Guide 2017 7

VENTANA anti-braf V600E (VE1) Mouse Monoclonal Primary Antibody VENTANA anti-braf V600E (VE1) Mouse Monoclonal Primary Antibody (VENTANA anti-braf V600E (VE1) antibody) is intended for the qualitative detection of BRAF V600E protein in formalin-fixed, paraffin-embedded tissue sections. VENTANA anti-braf V600E (VE1) antibody is ready to use on BenchMark ULTRA, XT and GX instruments with the OptiView DAB IHC Detection Kit and ancillary reagents. VENTANA anti-braf V600E (VE1) antibody is part of the VENTANA MMR IHC Panel which includes VENTANA anti- MLH1 (M1) Mouse Monoclonal Primary Antibody, VENTANA anti-pms2 (A16-4) Mouse Monoclonal Primary Antibody, VENTANA anti-msh2 (G219-1129) Mouse Monoclonal Primary Antibody and VENTANA anti-msh6 (SP93) Rabbit Monoclonal Primary Antibody. The VENTANA MMR IHC Panel is indicated for the detection of mismatch repair protein deficiency as a test for the identification of individuals at risk for Lynch syndrome in patients diagnosed with colorectal cancer (CRC), and, with BRAF V600E status, as an aid to differentiate between sporadic and probable Lynch syndrome CRC in the absence of MLH1 protein expression. These products should be interpreted by a qualified pathologist in conjunction with histological examination, relevant clinical information, and proper controls. Intended for in vitro diagnostic (IVD) use. Purpose of Interpretation Guide This guide is intended to aid pathologists in the clinical evaluation of formalin-fixed, paraffin-embedded (FFPE) colorectal carcinoma sections stained with the assays that comprise the VENTANA MMR IHC Panel in accordance with the proposed product labeling. Specifically this guide: Provides photographic images that illustrate the patterns and intensities of staining that may result from staining of colorectal carcinoma tissues with the assays that comprise the VENTANA MMR IHC Panel. Provides a reference for relating staining patterns and intensities to specific MMR biomarker clinical scores. Provides examples of challenging cases. Discusses other controls that may be used with the assay but are not provided by Ventana. 1016703EN Interpretation Guide 2017 8

Specimen Flow for Staining with the VENTANA MMR IHC Panel Tissue sample of colorectal carcinoma is taken from the patient, fixed in 10% neutral buffered formalin for 6-48 hours according to standard lab practices and embedded in paraffin. Sections 4-5 microns in thickness are mounted on positively charged slides. One section is stained with H&E. Repeat Staining NO Is the H&E slide acceptable? ( 50 viable tumor cells) YES For each assay in the VENTANA MMR IHC Panel: (1) One section is stained with a one of the following: VENTANA anti-mlh1 (M1) antibody VENTANA anti-pms2 (A16-4) antibody VENTANA anti MSH2 (G219-1129) antibody VENTANA anti-msh6 (SP93) antibody VENTANA anti-braf V600E (VE1) antibody (2) One section is stained with a negative reagent control antibody (corresponding to the species of the respective VENTANA MMR IHC Panel antibody) in the same staining run. 1016703EN Interpretation Guide 2017 9

Clinical Evaluation of MMR IHC Assays Evaluating Staining Patterns and Intensities Cells labeled with the IHC assays for the four MMR proteins (MLH1, PMS2, MSH2 and MSH6) are evaluated for presence or loss of the diaminobenzidine (DAB) signals. In colorectal carcinoma, the immunohistochemical staining of the four MMR proteins follows a nuclear staining pattern. In CRC cases with Lynch syndrome or somatic mutations, loss of any one of the four MMR proteins detected by the MMR IHC Assays is observed in the nuclei of tumor cells. The signal is classified as Intact or Loss based on nuclear localization only. Positive (Intact) signal is characterized by tumor cells that exhibit unequivocal nuclear staining of any intensity above background. Negative (Loss) signal intensity is characterized by an absence of any detectable signal or pale grey or tan nuclear discoloration in tumor cells. The DAB signal may be distributed homogeneously, having a uniform level of intensity throughout the neoplastic portions of the tumor or distributed heterogeneously having more than one intensity level. A species-matched negative control antibody is used to evaluate the presence of background in test samples and establish a staining intensity baseline. Each assay requires three serial tissue sections from each tissue specimen, one section for hematoxylin and eosin (H&E) staining, a second section for negative reagent control antibody staining, and a third section for staining with one of the MMR antibodies. A pre-qualified CRC tissue with a MMR status of intact may be used as a positive system-level control. If the H&E evaluation indicates that the patient specimen is inadequate (for example, if fewer than 50 viable tumor cells are present), then a new specimen should be obtained. Repeat staining of a specimen for a particular MMR IHC assay within the panel (e.g., VENTANA anti-mlh1 (M1) antibody) should be carried out on unstained slides if (1) the systemlevel positive control slide stained with that assay does not exhibit acceptable staining; (2) the case slide stained with the appropriate negative reagent control for that assay does not exhibit acceptable staining, or (3) if the case slide stained with that particular MMR IHC assay (e.g., VENTANA anti-mlh1 (M1) antibody) is not evaluable. If the last of these slides is not interpretable due to no staining in the internal positive control cells, artifacts, edge effects, necrosis, lack of tissue, or any other reason, then the slide cannot be used for clinical evaluation. If controls are acceptable and the VENTANA MMR assay-stained slide is evaluable, the slide can be evaluated by a trained pathologist as described in the Scoring Criteria. 1016703EN Interpretation Guide 2017 10

Specimen Flow for MMR IHC Assays THE FOLLOWING SHOULD BE EVALUATED FOR EACH SLIDE STAINED WITH A MMR IHC ASSAY FROM THE VENTANA MMR IHC PANEL Repeat staining that specimen slide. NO Is the negative reagent control slide acceptable? YES Repeat staining for that specimen slide. NO Is the specific MMR assay stained slide acceptable? YES A trained pathologist determines the clinical status for the specific MMR assay (e.g. VENTANA anti-mlh1 (M1) antibody) according to the scoring algorithm for the MMR assays that are part of the VENTANA MMR IHC Panel 1016703EN Interpretation Guide 2017 11

Morphology and Background Acceptability Criteria For each case slide stained with an MMR IHC assay, tissue morphology and background acceptability are assessed using the criteria described below in Table 1. Table 1: Morphology and Background Acceptability Criteria Morphology Background Acceptable Cellular elements of interest are visualized, allowing clinical interpretation of the stain Background does not interfere with clinical interpretation of the stain Unacceptable Cellular elements of interest are not visualized, compromising clinical interpretation of the stain Background interferes with ability to interpret the stain System-Level Control A known positive control tissue fixed and processed in the same manner as the patient specimens should be run for each set of test conditions and with every MMR IHC antibody staining procedure performed, to serve as a system-level control. The control tissue should be a fresh biopsy/surgical specimen prepared and fixed as soon as possible in a manner identical to patient specimens. This tissue is used to monitor all steps of specimen processing and staining. A tissue section fixed or processed differently from the test specimen can be used as a control for reagents and staining but not for fixation or tissue preparation. A positive tissue with moderate staining is more suitable for quality control than one that stains strongly; it can be used to detect minor levels of reagent degradation or out-of-specification issues that might be instrument-related. Positive control tissue may be on the same slide as the patient case or on a separate slide that is stained on the same run as the patient case. Pre-qualified CRC tissue with an IHC Clinical MMR status of Intact may be used as a positive system-level control. Alternatively pre-qualified normal colon tissue fixed and processed in the same manner as the patient tissue can also be used as a positive system-level control. Normal colon will stain positive for all MMR IHC antibodies. Acceptable staining with a system-level control will confirm that all the reagents for that particular MMR IHC assay (e.g., VENTANA anti- MLH1 (M1) antibody) were applied and the instrument functioned properly. The positive tissue control should be used only to monitor the correct performance of processed tissues, test reagents and instruments and not as an aid in formulating a specific diagnosis of patient samples. Pre-qualified CRC tissue with an IHC Clinical MMR status of Loss may be used as a negative system-level control. Since the MLH1, PMS2, MSH2, and MSH6 proteins are expressed in all tissues, a normal negative tissue control does not exist for these biomarkers. The negative tissue control should be used only to monitor the correct performance of 1016703EN Interpretation Guide 2017 12

processed tissues, test reagents and instruments and not as an aid in formulating a specific diagnosis of patient samples. Table 2: Staining Criteria for a Positive System-Level Control Intact Staining Pattern Acceptable Unequivocal nuclear staining in viable tumor cells, in the presence of internal positive controls (nuclear staining in lymphocytes, fibroblasts or normal epithelium in the vicinity of the tumor) Unacceptable Unequivocal loss of nuclear staining or focal weak equivocal nuclear staining in the viable tumor cells in the presence of internal positive controls 1016703EN Interpretation Guide 2017 13

Internal Positive Controls In colorectal carcinoma samples, unequivocal nuclear staining in lymphocytes, fibroblasts or normal epithelium in the vicinity of the tumor will serve as internal positive controls. Representative images of the internal control for the MMR IHC assays are shown below in Figure 2. H&E 10x Neg Ctrl 10x MLH1 10x MSH2 10x Figure 2: Internal Positive Controls Strong fibroblast staining (indicated by the blue arrows) is shown in a MLH1 loss status case and a MSH2 intact status case. 1016703EN Interpretation Guide 2017 14

CRC Case Criteria for Clinical Evaluation Table 3 summarizes the criteria for a CRC tissue slide to be evaluated for MMR status with any one of the MMR IHC assays. Table 3: Evaluable and Non-evaluable Criteria Clinical Interpretation Evaluable (all must be true) Staining Pattern 1) H&E has 50 viable tumor cells 2) Negative Reagent Control Slide is acceptable 3) Morphology is acceptable 4) Background is acceptable 5) System-level control is acceptable 6) Internal positive controls have unequivocal nuclear staining 1) H&E has < 50 viable tumor cells Not Evaluable (if one criteria in this section is true the slide staining should be repeated) 2) Negative Reagent Control Slide is unacceptable 3) Morphology is unacceptable 4) Background is unacceptable 5) System-level control is unacceptable 6) Internal positive controls do not have unequivocal nuclear staining 7) Interpretation is not possible due to tissue loss, tumor absence, artifacts and/or edge artifacts. 1016703EN Interpretation Guide 2017 15

Scoring Algorithm for MMR IHC Assays Clinical Status is assigned by a trained pathologist based on their evaluation of the presence or absence of specific staining with the four MMR IHC assays in the VENTANA MMR IHC Panel. A Clinical Status of Intact is assigned to cases with presence of nuclear staining for all four markers and a Clinical Status of Loss is assigned to cases with absence of nuclear staining in any one of the markers. Clinical interpretation of colorectal carcinoma cases stained with the MMR IHC assays should be based on the criteria noted in Table 4 below. Images of various Intact and Loss staining patterns are provided after the table. Table 4: Clinical Interpretation Criteria for the MMR IHC Assays Intact Protein Expression Unequivocal nuclear staining in viable tumor cells, in the presence of acceptable internal positive controls (nuclear staining in lymphocytes, fibroblasts or normal epithelium in the vicinity of the tumor) Loss of Protein Expression Unequivocal loss of nuclear staining or focal weak equivocal nuclear staining in the viable tumor cells in the presence of internal positive controls If unequivocal nuclear stain is absent in internal positive controls and/or background staining interferes with interpretation, the assay should be considered unacceptable and repeated. Punctate nuclear staining of tumor cells should be considered negative (loss). In cases with focal tumor cell staining, the intensity of the nuclear staining should be at least that of the internal positive controls along with the confluent /continuous staining of the nuclei in a few epithelial glands or nests for the case to be given a Clinical Status of Intact. In the absence of this, a Clinical Status of Loss is given to the case. 1016703EN Interpretation Guide 2017 16

Decision Tree for MMR IHC Assays Slides stained with MMR IHC assays should be evaluated using the Clinical Interpretation Criteria (Table 4) the approach is summarized in the decision tree below. THE FOLLOWING SHOULD BE EVALUATED FOR EACH MMR ASSAY IN THE VENTANA MMR IHC PANEL Is the negative reagent control slide acceptable? NO Repeat Staining or request a new specimen. YES Is the marker from the VENTANA MMR IHC Panel slide evaluable? YES Case is Intact for that VENTANA MMR IHC Marker YES Does the tumor have unequivocal nuclear staining? No Case is Loss for that VENTANA MMR IHC Marker Are all 4 markers in the VENTANA MMR IHC Panel Intact? Is one marker in the VENTANA MMR IHC Panel Loss? YES NO YES Case is Intact for VENTANA MMR IHC Status Case is Loss for VENTANA MMR IHC Status Case is Loss for VENTANA MMR IHC Status 1016703EN Interpretation Guide 2017 17

Cases with Intact MMR Status CRC Cases with Intact MMR protein expression An intact status staining result is characterized by the presence of detectable nuclear signal in the presence of appropriately stained internal controls. H&E 4x Neg Ctrl 4x MLH1 4x MLH1 10x Figure 3: MLH1 Intact Status tissue exhibits strong nuclear staining in the tumor cells in the presence of appropriately stained internal controls. 1016703EN Interpretation Guide 2017 18

H&E 4x Neg Ctrl 4x PMS2 4x PMS2 10x Figure 4: PMS2 Intact Status tissue exhibits strong nuclear staining in the tumor cells in the presence of appropriately stained internal controls. 1016703EN Interpretation Guide 2017 19

H&E 4x Neg Ctrl 4x MSH2 4x MSH2 10x Figure 5: MSH2 Intact Status tissue exhibits strong nuclear staining in the tumor cells in the presence of appropriately stained internal controls. 1016703EN Interpretation Guide 2017 20

H&E 4x Neg Ctrl 4x MSH6 4x MSH6 10x Figure 6: MSH6 Intact Status tissue exhibits strong nuclear staining in the tumor cells in the presence of appropriately stained internal controls. 1016703EN Interpretation Guide 2017 21

Cases with MMR Status of Loss CRC Cases with loss of MMR protein expression A loss status staining result is characterized by the absence of detectable nuclear signal in the presence of appropriately stained internal controls. H&E 4x Neg Ctrl 4x MLH1 4x MLH1 10x Figure 7: MLH1 Loss Status tissue exhibits no nuclear staining in the tumor cells in the presence of appropriately stained internal controls. 1016703EN Interpretation Guide 2017 22

H&E 4x Neg Ctrl 4x PMS2 4x PMS2 10x Figure 8: PMS2 Loss Status tissue exhibits no nuclear staining in the tumor cells in the presence of appropriately stained internal controls. 1016703EN Interpretation Guide 2017 23

H&E 4x Neg Ctrl 4x MSH2 4x MSH2 10x Figure 9: MSH2 Loss Status tissue exhibits no nuclear staining in tumor cells but weak cytoplasmic staining in the presence of appropriately stained internal controls. Cytoplasmic staining should be disregarded in the interpretation of status. 1016703EN Interpretation Guide 2017 24

H&E 4x Neg Ctrl 4x MSH6 4x MSH6 10x Figure 10: MSH6 Loss Status tissue exhibits no nuclear staining in the tumor cells in the presence of appropriately stained internal controls. 1016703EN Interpretation Guide 2017 25

Challenging Cases CRC cases are categorized as Intact or Loss for an MMR IHC assay according to the presence or absence of staining over the entire tumor area. The staining can vary in the level of intensity and this intensity may vary throughout the tumor; however, this does not impact MMR status. Some cases may be particularly challenging due to the following issues: Non-specific background Some specimens may exhibit non-specific background staining for reasons that are not well understood. For this reason, evaluation of a MMR IHC slide must include a comparison of the slide to the negative reagent control slide to determine the level of non-specific background staining. Cytoplasmic staining, if present, should be disregarded in MMR IHC interpretation. Focal Staining Some specimens may exhibit focal staining in the tumor cells and staining intensity may vary from weak to strong. Based on the MMR IHC scoring algorithm, focal weak equivocal nuclear staining in the viable tumor cells in the presence of internal positive controls should be categorized as Loss. Punctate Staining Some specimens may exhibit discrete punctate staining within a few nuclei of the tumor; the staining intensity may vary from weak to strong. This staining pattern should be ignored and if a case has only this type of staining pattern, it should be given a Clinical Status of Loss. Tissue or Staining Artifact Histologic artifacts originating from the sample processing and microtomy processes can also complicate the determination of MMR IHC Clinical Score. These artifacts may include, but are not limited to, fixation gradients and edge effects, DAB trapping, nuclear bubbling, lack of staining in some regions of the tissue, tearing or folding of the tissue, and loss of the tissue section. In some instances, repeat staining of new sections or acquisition of a new specimen may be required. Some challenging cases are shown on the following pages. 1016703EN Interpretation Guide 2017 26

Non-specific background staining H&E 4x Neg Ctrl 4x MSH2 4x MSH2 10x Figure 11: MSH2 Intact Status tissue exhibits strong nuclear staining in the presence of appropriately stained internal controls; cytoplasmic background staining is seen in the tumor cells which should be disregarded. 1016703EN Interpretation Guide 2017 27

H&E 4x Neg Ctrl 4x MSH2 4x MSH2 10x Figure 12: MSH2 Loss Status tissue exhibits pale tan cytoplasmic background in the tumor cells; however, no nuclear staining is present in the tumor in the presence of the appropriately stained internal positive controls. Cytoplasmic staining should be disregarded in the interpretation of status. 1016703EN Interpretation Guide 2017 28

Focal Staining H&E 4x Neg Ctrl 4x MSH6 4x MSH6 10x Figure 13: MSH6 Loss Status tissue exhibits focal weak equivocal nuclear staining in the viable tumor cells in the presence of appropriately stained internal positive controls. Also note that the intensity of the focal nuclear staining is less than that of the internal positive controls and not confluent around the gland. 1016703EN Interpretation Guide 2017 29

Punctate Staining H&E 10x Neg Ctrl 10x MLH1 10x MLH1 20x Figure 14: MLH1 Loss Status tissue tumor exhibits strong punctate staining in the nuclei of a few tumor cells however, there are no cells in the tumor that exhibit unequivocal nuclear staining in the presence of appropriately staining internal positive controls. 1016703EN Interpretation Guide 2017 30

Clinical Evaluation of VENTANA anti-braf V600E IHC Assay Evaluating Staining Patterns and Intensities In colorectal carcinoma, neoplastic cells labeled with the VENTANA anti-braf V600E (VE1) antibody are evaluated for the presence or absence of the diaminobenzidine (DAB) signal. The immunohistochemical staining in colorectal carcinoma follows a cytoplasmic staining pattern Positive signal is characterized by cells that exhibit unequivocal cytoplasmic staining; the intensity may range from weak to moderate. Negative signal intensity is characterized by an absence of any detectable signal. Negative cases may still exhibit pale grey or tan cytoplasmic discoloration. The signal may be distributed homogeneously, having a uniform level of intensity throughout the neoplastic portions of the tumor or distributed heterogeneously having more than one intensity level. An isotype-matched negative control antibody is used to evaluate the presence of background in test samples and establish a staining intensity baseline. The VENTANA anti-braf V600E (VE1) IHC assay requires one serial tissue section for hematoxylin and eosin (H&E) staining, a second serial tissue section for negative reagent control antibody staining and a third serial tissue section for VENTANA anti-braf V600E (VE1) antibody staining. A CRC that is positive for BRAF V600E mutation by VENTANA anti- BRAF V600E (VE1) antibody IHC may be used as a system-level control. If the H&E evaluation indicates that the patient specimen is inadequate (for example, if fewer than 50 viable tumor cells are present), then a new specimen should be obtained. Repeat staining of a specimen should be carried out on unstained slides if (1) the system-level positive control slide does not exhibit acceptable staining; (2) the negative reagent control antibody case slide does not exhibit acceptable staining; or (3) the BRAF V600E stained case slide is not evaluable. If the last of these slides is not interpretable due to artifacts, edge effects, necrosis, lack of tissue, or any other reason, then the slide cannot be used for clinical evaluation. If controls are acceptable and the VENTANA anti-braf V600E (VE1) antibody IHC slide is evaluable, the slide can be evaluated by a trained pathologist as described in the Scoring Criteria. 1016703EN Interpretation Guide 2017 31

Specimen Flow for VENTANA anti-braf V600E IHC Assay THE FOLLOWING SHOULD BE EVALUATED FOR EACH SLIDE STAINED WITH VENTANA ANTI-BRAF V600E (VE1) ANTIBODY FROM THE VENTANA MMR IHC PANEL Repeat staining run. NO Is the negative reagent control slide acceptable? YES Repeat staining run. NO Is the VENTANA anti-braf V600E (VE1) antibody stained specimen slide acceptable? YES BRAF V600E status is determined by a trained pathologist according to the VENTANA anti-braf V600E (VE1) antibody clinical scoring algorithm that is part of the VENTANA MMR IHC Panel 1016703EN Interpretation Guide 2017 32

Morphology and Background Acceptability Criteria Tissue morphology and background acceptability are assessed for each patient case using the criteria described below in Table 5. Table 5: Morphology and Background Acceptability Criteria Morphology Background Acceptable Cellular elements of interest are visualized, allowing clinical interpretation of the stain Background does not interfere with clinical interpretation of the stain Unacceptable Cellular elements of interest are not visualized, compromising clinical interpretation of the stain Background interferes with ability to interpret the stain System-level Control A known system level control tissue fixed and processed in the same manner as the patient specimens should be run with every VENTANA anti-braf V600E (VE1) antibody staining protocol performed. The control tissue should be a fresh biopsy/surgical specimen prepared and fixed as soon as possible in a manner identical to patient specimens. This tissue is used to monitor all steps of specimen processing and staining. A tissue section fixed or processed differently from the test specimen can be used as a control for reagents and staining but not for fixation or tissue preparation. A positive tissue with moderate staining is more suitable for quality control than one that stains strongly; it can be used to detect minor levels of reagent degradation or out-of-specification issues that might be instrument-related. System-level control tissue may be run on the same slide as the patient case or on a separate slide run at the same time as the patient case. A positive system-level control would be a pre-qualified case of CRC that is positive for BRAF V600E mutation by VENTANA anti-braf V600E (VE1) antibody IHC. The staining of the CRC tissue case that has the BRAF V600E mutation will confirm that all the reagents were applied and the instrument functioned properly. The positive tissue control should be used only to monitor performance; it should not be used to aid the clinical diagnosis of patient samples. A negative system-level control would be a pre-qualified case of CRC that is negative for VENTANA anti-braf V600E (VE1) antibody IHC negative. The negative tissue control should be used only to monitor performance; it should not be used to aid the clinical diagnosis of patient samples. Control tissue on each VENTANA anti-braf V600E (VE1) antibody stained slide will be judged as acceptable or unacceptable by the reviewing pathologists according to criteria outlined in the Table 6. 1016703EN Interpretation Guide 2017 33

Table 6: Staining Criteria for a Positive System-Level Control CRC tissue Acceptable Unequivocal cytoplasmic staining of any intensity in viable tumor cells above background. Unacceptable No staining or equivocal cytoplasmic staining in viable tumor cells. Fibroblasts and lymphocytes in normal epithelium No staining or equivocal cytoplasmic staining in fibroblasts and lymphocytes in normal epithelium. Unequivocal cytoplasmic staining in the fibroblasts and lymphocytes in the normal epithelium. Scoring Algorithm for the VENTANA anti-braf V600E IHC Assay VENTANA anti-braf V600E (VE1)antibody IHC status is assigned by a trained pathologist based on his or her evaluation of the intensity of specific staining for VENTANA anti-braf V600E (VE1) as illustrated in Table 7 below. A Positive status is assigned to cases with positive staining and a Negative Status is assigned to cases with absent staining. Clinical interpretation of colorectal carcinoma cases stained with VENTANA anti-braf V600E (VE1) antibody should be based on the criteria noted in the Table 7. Images of various Negative and Positive staining patterns are provided after Table 7. Table 7: Clinical Interpretation Criteria for the VENTANA anti-braf V600E (VE1) IHC Assay Positive for BRAF V600E mutation Unequivocal cytoplasmic staining of any intensity in viable tumor cells above background. Negative for BRAF V600E mutation No staining or equivocal cytoplasmic staining in viable tumor cells. Note: Nuclear staining, weak to strong staining of isolated viable tumor cells/or small tumor clusters should be considered negative. 1016703EN Interpretation Guide 2017 34

Decision Tree THE FOLLOWING SHOULD BE EVALUATED FOR VENTANA anti-braf V600E (VE1) ANTIBODY IN THE VENTANA MMR IHC PANEL Is the negative reagent control slide acceptable? NO Repeat Staining or request a new specimen. YES Is the VENTANA anti- BRAF V600E (VE1) antibody slide evaluable? YES Case is Positive for BRAF V600E. YES Does the tumor have unequivocal cytoplasmic staining? No Case is Negative for BRAF V600E. 1016703EN Interpretation Guide 2017 35

Cases Negative for BRAF V600E CRC Cases with BRAF V600E Negative Status A negative staining result is characterized by an absence of any detectable signal. Negative cases may still exhibit pale grey or tan cytoplasmic discoloration. H&E 4x Neg Ctrl 4x BRAF V600E 4x BRAF V600E 10x Figure 15: BRAF V600E Negative Status tissue exhibits no cytoplasmic staining in the tumor cells. 1016703EN Interpretation Guide 2017 36

H&E 4x Neg Ctrl 4x BRAF V600E 4x BRAF V600E 10x Figure 16: BRAF V600E Negative Status tissue exhibits a light cytoplasmic blush. 1016703EN Interpretation Guide 2017 37

Cases Positive for BRAF V600E CRC Cases with BRAF V600E Positive Status A positive staining result is characterized by cells that exhibit unequivocal cytoplasmic staining; the intensity may range from weak to moderate. H&E 4x Neg Ctrl 4x BRAF V600E 4x BRAF V600E 10x Figure 17: BRAF V600E Positive Status tissue exhibits weak cytoplasmic staining. 1016703EN Interpretation Guide 2017 38

H&E 4x Neg Ctrl 4x BRAF V600E 4x BRAF V600E 10x Figure 18: BRAF V600E Positive Status tissue exhibits a weak to moderate cytoplasmic staining. 1016703EN Interpretation Guide 2017 39

H&E 4x Neg Ctrl 4x BRAF V600E 4x BRAF V600E 10x Figure 19: BRAF V600E Positive Status tissue exhibits moderate cytoplasmic staining. 1016703EN Interpretation Guide 2017 40

H&E 4x Neg Ctrl 4x BRAF V600E 4x BRAF V600E 10x Figure 20: BRAF V600E Positive Status tissue exhibits strong cytoplasmic staining. 1016703EN Interpretation Guide 2017 41

Challenging Cases Some cases may be particularly challenging due to the following issues: Non-specific background Some specimens may exhibit non-specific background staining for reasons that are not well understood. VENTANA anti-braf V600E (VE1) antibody was found to occasionally exhibit cytoplasmic background staining in smooth muscle and nuclear staining in normal colon epithelial cells, enterocytes, Leydig cells of testis, adrenal gland, pituitary gland and some tumor cells; however, such cases should not be considered as positive for the BRAF V600E mutation. In addition, this antibody also stains cilia in lung. For this reason, evaluation of a VENTANA anti-braf V600E (VE1) antibody stained slide must include a comparison to the negative control slide to determine the level of non-specific background staining. Nuclear Staining Some specimens may only exhibit nuclear staining. Based on the VENTANA anti-braf V600E (VE1) antibody IHC scoring algorithm, these cases should be considered negative. Tissue or Staining Artifact Histologic artifacts originating from the sample processing and microtomy processes can also complicate the determination of VENTANA anti-braf V600E (VE1) antibody IHC Clinical Status. These artifacts may include, but are not limited to, fixation gradients and edge effects, DAB trapping, nuclear bubbling, lack of staining in some regions of the tissue, tearing or folding of the tissue, and loss of the tissue section. In some instances, repeat staining of new sections or acquisition of a new specimen may be required. Heterogeneous Staining Some cases may show only focal, but unequivocal cytoplasmic staining. These cases are challenging and additional testing may be considered (Fig 25). Some challenging cases are shown on the following pages. 1016703EN Interpretation Guide 2017 42

Non-Specific Background Staining H&E 4x Neg Ctrl 4x BRAF V600E 4x BRAF V600E 10x Figure 21: BRAF V600E Negative Status tissue exhibits a weak cytoplasmic blush throughout the entire tumor. 1016703EN Interpretation Guide 2017 43

Non-Specific High Background Staining H&E 4x Neg Ctrl 4x BRAF V600E 4x BRAF V600E 10x Figure 22: BRAF V600E Positive Status tissue exhibits a high non-specific background outside of the tumor cells. 1016703EN Interpretation Guide 2017 44

Nuclear Staining H&E 4x Neg Ctrl 4x BRAF V600E 4x BRAF V600E 10x Figure 23: BRAF V600E Negative Status tissue exhibits no cytoplasmic staining in the tumor cells. Moderate nuclear staining is present in some cells, however, based on the interpretation criteria only cytoplasmic staining should be used in determining the status for BRAF V600E. 1016703EN Interpretation Guide 2017 45

H&E 4x Neg Ctrl 4x BRAF V600E 4x BRAF V600E 10x Figure 24: BRAF V600E Positive Status tissue exhibits unequivocal strong cytoplasmic staining within the tumor cells. Moderate nuclear staining is present in some cells, however, based on the interpretation criteria only cytoplasmic staining should be used in determining the status for BRAF V600E. 1016703EN Interpretation Guide 2017 46

Focal Staining BRAF V600E 2x H&E 4x Neg Ctrl 4x BRAF V600E 4x BRAF V600E 10x Figure 25: BRAF V600E Positive Status tissue exhibits heterogeneous BRAF V600E staining intensity. Weak equivocal cytoplasmic staining is present in the majority of the tumor, with focal moderate to strong unequivocal cytoplasmic staining. 1016703EN Interpretation Guide 2017 47

VENTANA MMR IHC Panel Case Examples H&E 10x Neg Ctrl 10x MLH1 10x PMS2 10x MSH2 10x MSH6 10x Figure 26: MMR Proficient CRC this case exhibits proficient DNA mismatch repair status (pmmr) for the four MMR markers in the VENTANA MMR IHC Panel. 1016703EN Interpretation Guide 2017 48

H&E 10x Neg Ctrl 10x MLH1 10x PMS2 10x MSH2 10x MSH6 10x BRAF V600E 10x Figure 27: Sporadic Colon Cariconoma this case exhibits deficient DNA mismatch repair status (dmmr) due to loss of MLH1 and PMS2 with intact MSH2 and MSH6. BRAF V600E is positive for this case. 1016703EN Interpretation Guide 2017 49

H&E 10x Neg Ctrl 10x MLH1 10x PMS2 10x MSH2 10x MSH6 10x BRAF V600E 10x Figure 28: Probable Lynch Syndrome this case exhibits deficient DNA mismatch repair status (dmmr) due to MSH2 and MSH6 loss, with intact MLH1 and PMS2. BRAF V600E is negative for this case. 1016703EN Interpretation Guide 2017 50

H&E 10x Neg Ctrl 10x MLH1 10x PMS2 10x MSH2 10x MSH6 10x BRAF V600E 10x Figure 29: Probable Lynch Syndrome this case exhibits deficient DNA mismatch repair status (dmmr) due to MLH1 and PMS2 loss, with intact MSH2 and MSH6, BRAF V600E is negative for this case. 1016703EN Interpretation Guide 2017 51

Cut Slide Stability Ventana recommends that sections approximately 4 µm in thickness should be cut and mounted on positively charged slides. Slides should be stained within 8 weeks of preparation. Day 0 Week 4 Week 10 MLH1 Day 0 Week 4 Week 8 PMS2 Day 0 Week 4 Week 10 MSH2 Day 0 Week 4 Week 10 MSH6 Day 0 Week 4 Week 10 BRAF V600E Figure 30: Cut Slide Stability Staining. This panel exhibits cases that have an intact status for MLH1, PMS2, MSH2 and MSH6 and a Positive status for BRAF V600E for the time points indicated in the figure. 1016703EN Interpretation Guide 2017 52

References 1. Ferlay J, S.I., Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray, F., GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. 2012, Lyon, France: International Agency for Research on Cancer; 2013. 2. Howlader N, N.A., Krapcho M, Garshell J, Miller D, Altekruse SF, Kosary CL, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA (eds). SEER Cancer Statistics Review, 1975-2012.2015, National Cancer Institute. Bethesda, MD. 3. Lynch, H.T. and A. de la Chapelle, Hereditary colorectal cancer. N Engl J Med, 2003. 348(10): p. 919-32. 4. Aaltonen, L.A., et al., Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease. N Engl J Med, 1998. 338(21): p. 1481-7. 5. Giardiello, F.M., et al., Guidelines on genetic evaluation and management of Lynch syndrome: a consensus statement by the US Multi-society Task Force on colorectal cancer. Am J Gastroenterol, 2014. 109(8): p. 1159-79. 6. Stoffel, E., et al., Calculation of risk of colorectal and endometrial cancer among patients with Lynch syndrome. Gastroenterology, 2009. 137(5): p. 1621-7. 7. Gill, S., et al., Isolated loss o PMS2 Expression in colorectal cancers: fequency,patient age, and familial aggregation. Clinical Cancer Resarch, 2005. 11(18):p.6466-71. 8. Buchanan, D.D., et al., Tumor Mismatch Repair Immunohistochemistry and DNA MLH11 Methylation Testing of Patients With Endometrial Cancer Diagnosed at Age Younger Than 60 Years Optimizes Triage for Population-Level Germline Mismatch Repair Gene Mutation Testing. J of Clincal Oncology, 2014. 32(2):p.90-100. 1016703EN Interpretation Guide 2017 53

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